Restriction Enzyme Digestion Of Duck Enteritis Virus Genomic DNA And Analysis On CHEF MAPPER, And Construction Of UL41 Mutant

After propagating in chicken embryo fibroblast (CEF), high titer Duck Enteritis Virus (C-KCE strain) was collected to extract viral DNA, then the viral DNA abtained was degested by restriction enzyme. CHEF MAPPER was employed to analysis digested products, and results showed the whole DEV genome located between 145kb-194kb and the DNA sample was highly purified and ready to be used for genomic library construction.CEF was infected by DEV with dilution of MOI 1 and MOI 0.01 to study virus titer in the cell culture fluid and its growth and replication status, as the results showed DEV could growth well in CEP and TCID50 was 10-7.55 post infection 48h.According to the UL41 genome of DEV, the specific primers were designed to amplify UL41 genome by PCR, and then the UL41 Mutant was amplified by PCR used mutation primer. UL41 Mutant DEV was constructed successfully with Red homologous-recombination to transfect the CEP, and the results of immunofluorescence assay dicated that UL41 Mutant DEV can proliferate and express proteins normally. This indicated that UL41 is not essential for virus replication.